Du Weijie, Pan Zhenwei, Chen Xu, Wang Leimin, Zhang Ying, Li Shuang, Liang Haihai, Xu Chaoqian, Zhang Yong, Wu Yanping, Shan Hongli, Lu Yanjie
Department of Pharmacology (Key Laboratory of Cardiovascular Medicine Research, Ministry of Education; State-Province Key Laboratories of Biomedicine-Pharmaceutics of China), Harbin Medical University, Harbin, Heilongjiang, People's Republic of China.
Cell Physiol Biochem. 2014;34(3):955-65. doi: 10.1159/000366312. Epub 2014 Aug 26.
Several studies have confirmed the role of microRNAs in regulating ischemia/reperfusion-induced cardiac injury (I/R-I). MiR-17-5p has been regarded as an oncomiR in the development of cancer. However, its potential role in cardiomyocytes has not been exploited. The aim of this study is to investigate the role of miR-17-5p in I/R-I and the underlying mechanism through targeting Stat3, a key surviving factor in cardiomyocytes.
MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) assay was used to detect the cell viability. ELISA and TUNEL were performed to measure apoptosis of neonatal rat ventricular cardiomyocytes (NRVCs). Infarct area was estimated by TTC (triphenyltetrazolium chloride) and Evans blue staining. Western blot analysis was employed to detect the Stat3 and p-Stat3 levels and real-time RT-PCR was used to quantify miR-17-5p level.
The miR-17-5p level was significantly up-regulated in I/R-I mice and in NRVCs under oxidative stress. Overexpression of miR-17-5p aggravated cardiomyocyte injury with reduced cell viability and enhanced apoptotic cell death induced by H2O2, whereas inhibition of miR-17-5p by its antisense AMO-17-5p abrogated the deleterious changes. Moreover, the locked nucleic acid-modified antisense (LNA-anti-miR-17-5p) markedly decreased the infarct area and apoptosis induced by I/R-I in mice. Furthermore, overexpression of miR-17-5p diminished the p-Stat3 level in response to H2O2. The results from Western blot analysis and luciferase reporter gene assay confirmed Stat3 as a target gene for miR-17-5p.
Upregulation of miR-17-5p promotes apoptosis induced by oxidative stress via targeting Stat3, accounting partially for I/R-I.
多项研究已证实微小RNA在调节缺血/再灌注诱导的心脏损伤（I/R-I）中的作用。MiR-17-5p在癌症发展中被视为一种癌基因。然而，其在心肌细胞中的潜在作用尚未得到充分研究。本研究的目的是通过靶向心肌细胞中的关键存活因子Stat3，探讨miR-17-5p在I/R-I中的作用及其潜在机制。
采用MTT（3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐）法检测细胞活力。进行ELISA和TUNEL检测新生大鼠心室肌细胞（NRVCs）的凋亡情况。通过TTC（氯化三苯基四氮唑）和伊文思蓝染色评估梗死面积。采用蛋白质免疫印迹分析检测Stat3和p-Stat3水平，实时RT-PCR用于定量miR-17-5p水平。
在I/R-I小鼠和氧化应激下的NRVCs中，miR-17-5p水平显著上调。miR-17-5p的过表达加重了心肌细胞损伤，降低了细胞活力，并增强了H2O2诱导的凋亡细胞死亡，而其反义寡核苷酸AMO-17-5p抑制miR-17-5p则消除了这些有害变化。此外，锁核酸修饰的反义寡核苷酸（LNA-anti-miR-17-5p）显著降低了I/R-I诱导的小鼠梗死面积和凋亡。此外，miR-17-5p的过表达降低了H2O2刺激下的p-Stat3水平。蛋白质免疫印迹分析和荧光素酶报告基因检测结果证实Stat3是miR-17-5p的靶基因。
miR-17-5p的上调通过靶向Stat3促进氧化应激诱导的凋亡，这部分解释了I/R-I的发生机制。
